Successful production of recombinant proteins has been accomplished with eukaryotic hosts. The most prominent examples are yeasts like Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus awamori or Trichoderma reesei, or mammalian cells like e.g. CHO cells. While the production of some proteins is readily achieved at high rates, many other proteins are only obtained at comparatively low levels.
The heterologous expression of a gene in a host organism usually requires a vector allowing stable transformation of the host organism. A vector would provide the gene with a functional promoter adjacent to the 5′ end of the coding sequence. The transcription is thereby regulated and initiated by this promoter sequence. Most promoters used up to date have been derived from genes that code for proteins that are usually present at high concentrations in the cell.
EP0103409A2 discloses the use of yeast promoters associated with expression of specific enzymes in the glycolytic pathway, i.e. promoters involved in expression of pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, phosphor-glycerate mutase, hexokinase 1 and 2, glucokinase, phosphofructose kinase, aldolase and glycolytic regulation gene.
WO 97/44470 describes yeast promoters from Yarrowia lipolytica for the translation elongation factor 1 (TEF1) protein and for the ribosomal protein S7 that are suitable for heterologous expression of proteins in yeast, and EP1951877A1 describes the use of the P. pastoris TEF1 promoter for the production of heterologous proteins.
WO2005003310 provides methods for the expression of a coding sequence of interest in yeast using a promoter of the glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate mutase from oleaginous yeast Yarrowia lipolytica. 
Promoter sequences derived from genes involved in the methanol metabolic pathway of Pichia pastoris are disclosed in U.S. Pat. No. 4,808,537 and U.S. Pat. No. 4,855,231 (alcohol oxidase AOX1, AOX2) and U.S. Pat. No. 6,730,499B1 (formaldehyde dehydrogenase FLD1). US20080153126A1 includes mutant promoter sequences based on the AOX1 promoter.
The AOX1 promoter is induced only in response to methanol and repressed by other carbon sources, such as glucose or ethanol. Methanol has the disadvantage that it is unsuitable for use in the production of certain products, since it is potentially hazardous for its toxicity and flammability. Therefore, alternatives to the AOX1 promoter are sought.
Vassileva et al. (J. Biotechnol. (2001) 88: 21-35) describe the use of the GAP promoter to express HBsAg in P. pastoris, using multicopy expression cassettes as an alternative to the AOX1 promoter. The constitutive system was proposed for continuous culture to permit maintenance of the cells in mid-exponential phase.
Promoters used in Pichia pastoris are either tightly regulated (like pAOX or pFLD) being active on specific substrates such as methanol, or they are constitutively active in many different conditions, media and substrates. Among the constitutive ones, especially the GAP and the TEF promoters have been described to be strong, and useful for recombinant protein production.
However, it was shown that the activity of both constitutive promoters is not constantly strong during a fed-batch production process. Especially in the later phases of the process, when cell growth rates are slow, also the activity of the promoters in getting low, thus limiting expression levels of the gene of interest (GOI) and production yields (Stadlmayr et al. 2010. J Biotechnol. 150: 519-529).
Selection of suitable promoters is not intuitive or rational, as even highly abundant glycolytic enzymes such as enolase (ENO), triose phosphate isomerase (TPI) or glucose-6-phosphate isomerase (PGI) do not have promoters that are as strong as pGAP and pTEF (Stadlmayr et al. 2010. J Biotechnol. 150: 519-529; Gasser et al. 2010. Metabolic Engineering 12:573-580).
Qin et al. (Applied and Environmental Microbiology (2011) 3600-3608) describe a GAP promoter library and various mutants with varying activities.
It is desirable to provide improved recombinant eukaryotic cell lines to produce fermentation products that can be isolated with high yields. Therefore, it is the object of the present invention to provide for alternative regulatory elements suitable for recombinant production methods, which are simple and efficient.